Histological Staining Area Quantification 1.0
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Workflow Type: Galaxy
This workflow performs colour deconvolution and automated thresholding to quantify stained area in brightfield histological images (MT, IHC). It accepts whole slides or pre-cropped ROIs.
Associated Tutorial
This workflows is part of the tutorial Quantitative Analysis of Histological Staining Using Color Deconvolution, available in the GTN
Features
- Includes Galaxy Workflow Tests
- Includes a Galaxy Workflow Report
- Uses Galaxy Workflow Comments
Thanks to...
Workflow Author(s): Diana Chiang Jurado
Tutorial Author(s): Diana Chiang Jurado
Tutorial Contributor(s): Leonid Kostrykin
Funder(s): University of Freiburg
Grants(s): NFDI4Bioimage
Inputs
| ID | Name | Description | Type |
|---|---|---|---|
| ROI image for staining analysis | ROI image for staining analysis | Brightfield histological image (whole slide or pre-cropped ROI) for staining quantification. Compatible stainings include Masson's Trichrome and IHC. Accepted format: TIFF. |
|
Steps
| ID | Name | Description |
|---|---|---|
| 1 | Sample ID | Add Sample ID column name . toolshed.g2.bx.psu.edu/repos/bgruening/text_processing/tp_text_file_with_recurring_lines/9.5+galaxy3 |
| 2 | header for total area | toolshed.g2.bx.psu.edu/repos/bgruening/text_processing/tp_text_file_with_recurring_lines/9.5+galaxy3 |
| 3 | Stain intensity normalization (CLAHE) | Normalizes pixel intensity across images using CLAHE (Contrast Limited Adaptive Histogram Equalization) to reduce staining variability before color deconvolution. toolshed.g2.bx.psu.edu/repos/imgteam/2d_histogram_equalization/ip_histogram_equalization/0.25.2+galaxy0 |
| 4 | Show image info | toolshed.g2.bx.psu.edu/repos/imgteam/image_info/ip_imageinfo/8.0.0+galaxy0 |
| 5 | Color Deconvolution | Performs colour deconvolution using the H-E-DAB (HED) transformation, separating the image into individual stain channels. Channel 1 = Hematoxylin, Channel 2 = Eosin/target stain, Channel 3 = Residuals. Higher pixel intensity indicates lower stain concentration. toolshed.g2.bx.psu.edu/repos/imgteam/color_deconvolution/ip_color_deconvolution/0.9+galaxy0 |
| 6 | Width Extraction | Grep1 |
| 7 | Height Extraction | Grep1 |
| 8 | Split Image Channels for Staining Detection | Splits multi-channel image into single-channel images. Extracts the target stain channel from the split image collection. Default index: 1 (Channel 2). Verify this corresponds to your stain of interest before running the workflow. The pixel data type is preserved. toolshed.g2.bx.psu.edu/repos/imgteam/split_image/ip_split_image/2.3.5+galaxy1 |
| 9 | Text transformation | toolshed.g2.bx.psu.edu/repos/bgruening/text_processing/tp_sed_tool/9.5+galaxy3 |
| 10 | Text transformation | toolshed.g2.bx.psu.edu/repos/bgruening/text_processing/tp_sed_tool/9.5+galaxy3 |
| 11 | Collection: Extract Stain Channel from Sub-Collections | This step extracts the second image (usually the relevant stain channel) from each sub-collection in a collection of collections. Please verify the selected channel is correct before proceeding. The current index is set to extract dataset 2. __EXTRACT_DATASET__ |
| 12 | Paste | Paste1 |
| 13 | Threshold Stain Channel Collection | Applies a threshold to the selected stain channel to segment stained areas for quantification. Tool only process tiff files with 1 channel. toolshed.g2.bx.psu.edu/repos/imgteam/2d_auto_threshold/ip_threshold/0.25.2+galaxy0 |
| 14 | Compute | toolshed.g2.bx.psu.edu/repos/devteam/column_maker/Add_a_column1/2.1+galaxy0 |
| 15 | Generate ROIs of stained region | This step detects CD11b positive regions from the binary mask and generates polygon ROIs for each stained blob. ROIs can be uploaded to OMERO for visual validation of the threshold. toolshed.g2.bx.psu.edu/repos/imgteam/imagej2_analyze_particles_binary/imagej2_analyze_particles_binary/20240614+galaxy1 |
| 16 | Extract Image Features | Extracts quantitative features (e.g., mean intensity) from the thresholded image to support staining analysis. toolshed.g2.bx.psu.edu/repos/imgteam/2d_feature_extraction/ip_2d_feature_extraction/0.25.2+galaxy1 |
| 17 | Cut | Cut1 |
| 18 | Prepare Header Source Dataset | Extracts a dataset that will be used to obtain the header row for the final concatenated tabular file in a subsequent step. __EXTRACT_DATASET__ |
| 19 | Extract | Show tail1 |
| 20 | Extract element identifiers | toolshed.g2.bx.psu.edu/repos/iuc/collection_element_identifiers/collection_element_identifiers/0.0.3 |
| 21 | Collapse Collection | toolshed.g2.bx.psu.edu/repos/nml/collapse_collections/collapse_dataset/5.1.0 |
| 22 | Header Extraction | Extracting header from source dataset to use in the final tabular file. Show beginning1 |
| 23 | Collapse Collection into one file | Allows to collapse a collection of tabular files with the individual samples quantification into one tabular file. toolshed.g2.bx.psu.edu/repos/nml/collapse_collections/collapse_dataset/5.1.0 |
| 24 | Concatenate datasets | toolshed.g2.bx.psu.edu/repos/bgruening/text_processing/tp_cat/9.5+galaxy3 |
| 25 | Paste header with Sample ID | Combined the Sample ID with the header of the remaining columns. Paste1 |
| 26 | Paste Sample Names with Tabular Results | This step joins a list of sample names with their corresponding quantification results. It pastes both files side by side, ensuring that each sample name is aligned with the correct row of the quantification table. Paste1 |
| 27 | Tabular: Staining Feature Results | This final output file contains the merged measurement results for all samples, and can be used for further statistical analysis. cat1 |
| 28 | Paste | Paste1 |
| 29 | Percent area computation | Computes the proportion of positive signal area relative to total image area, expressed as percentage. toolshed.g2.bx.psu.edu/repos/devteam/column_maker/Add_a_column1/2.1+galaxy0 |
Outputs
| ID | Name | Description | Type |
|---|---|---|---|
| Deconvolved Image | Deconvolved Image | n/a |
|
| Collection: Individual Deconvolved Channels | Collection: Individual Deconvolved Channels | n/a |
|
| Selected Stain Channel | Selected Stain Channel | n/a |
|
| Selected Stain Channel Thresholded | Selected Stain Channel Thresholded | n/a |
|
| ROIs | ROIs | n/a |
|
| ROIs Image | ROIs Image | n/a |
|
| Collection of Tabular: Staining Quantification Results | Collection of Tabular: Staining Quantification Results | n/a |
|
| Tabular File: Staining Feature Results | Tabular File: Staining Feature Results | n/a |
|
Version History
master
Creators and Submitter
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Activity
Views: 7 Downloads: 1 Runs: 0
Created: 15th Jun 2026 at 16:08
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